Project Description:

ATHELAS: A Modular Screening Platform for Stabilized Antimicrobial Peptides Expressed in Nicotiana benthamiana

Abstract

Antimicrobial resistance is one of the ten most severe global health threats to humanity, according to the WHO. Pathogenic bacteria are constantly acquiring resistance to further antibiotics, leading to the emergence of multi-drug-resistant and therefore untreatable strains with the drugs available. Antimicrobial peptides (AMPs), which are part of the innate immune response of eukaryotes and prokaryotes, represent a class of new potential antibacterial drugs. However, the medical applicability of unmodified AMPs is limited by their low in vivo stability. Therefore, we invented ATHELAS: an AMP-thioknot herbal expression and low effort activity screening platform. In our project, we use highly stable cyclic peptides from plants called cyclotides as a scaffold to stabilize AMPs. We developed a convenient platform to screen the properties of arbitrary stabilized peptides. Our platform includes an array of fast cloning, transient expression in Nicotiana benthamiana, efficient extraction and purification, and antibacterial activity testing.

Antimicrobial Resistance: A Major Threat to Global Health

In 1928, the British bacteriologist Alexander Fleming made a discovery that would lead to a milestone in modern medicine. He observed the mold Penicillium notatum inhibiting the growth of several pathogenic bacterial strains ³⁸ . The responsible compound turned out to be penicillin, the first natural antibiotic to be discovered. Soon after its discovery, manufacturing as a drug began in the USA and it was available to the public in large amounts by 1945 ³⁸ . Penicillin became one of many antibiotics discovered and developed in the years following ² . Though impossible to accurately quantify, antibiotics are believed to have saved millions of human lives until today, including many wounded soldiers during the Second World War ² . The significance of Flemming's discovery is illustrated, as the course of history might have been altered drastically since influential historical personalities like Churchill are believed to have been cured with antibiotics in their lives ² . It is therefore needless to say that antibiotics are one of the key discoveries in modern human history, which was awarded the Nobel prize in 1945 ³⁸ .

Despite this development in the last century, antibiotics are constantly losing the potential to treat bacterial infections. The main reason for this is that bacteria can acquire resistance against antibiotics. These antibiotic resistances (ABR) are mostly conferred by special resistance genes, which are located on plasmids. Plasmids are circular extrachromosomal DNA, which can be inherited and transferred between bacteria, thereby leading to a positive selection of bacteria possessing the resistance genes. Due to the spread of such genes, pathogenic Staphylococcus aureus (S. aureus) strains became resistant to penicillin quickly. In 1946, 14 % of S. aureus hospital infections were reported to be immune to penicillin. That number would rise to 80 % in 1953 ⁷ . An investigation of infections with antibiotic-resistant bacteria in the EU, published in 2019, found that infections with such pathogens rose to an incidence of 131 bacterial infections per 100,000 EU inhabitants in 2015 ⁸ . In 2015 (in the EU), antibiotic-resistant bacterial infections accounted for roughly the same burden as the infectious diseases influenza, tuberculosis, and HIV together, measured in disability-adjusted life years ⁸ . Even pathogenic strains resistant against third-generation cephalosporins and carbapenems, which have been declared critically important, reserve antimicrobials by the World Health Organization (WHO) ⁴² , have been increasing significantly from 2007 to 2015 ( figure 1 ) ⁸ .

Death toll in the EU in 2007 and 2015 of the four most deadly antibiotic-resistant bacteria and world-wide approvals of antibiotic-based drugs from 1980 to 2019 — Figure 1: (A) Death tolls in the EU in 2007 and 2015, attributable to the four most deadly antibiotic-resistant bacteria ⁸ ; (B) Antibiotic-based drugs approved worldwide from 1980 to 2019 ³⁰ .

Clearly, the situation is critical. WHO predicts that by 2050 death toll due to drug-resistant pathogens could rise to 10 million people per year ⁴³ , and has declared AMR one of the ten greatest threats to global human health ²³ . Since AMR is mainly caused by mis- and overuse of antibiotics in different sectors, one solution to this threat could be better coordination and interplay of these different sectors. This approach is called “One Health”, aiming to limit the spread of resistant pathogenic bacteria by restricting and coordinating the use of antimicrobials in humans, agriculture, and the environment, thereby preventing the transmission of AMR between these sectors ²⁹ . On the other hand, new antibiotics could be developed to combat pathogens already resistant to formerly used antibiotics. This approach was successful in the past when 30 new antibiotics were approved for use in humans from 1983 to 1992 ⁹ . However, the number of approvals has declined since then. From 2010 to 2021, only 17 new antibiotic drugs have been approved, all of them being derivatives of already in-use antibiotics ⁹ . This is because developing new antibiotics is economically infeasible for pharmaceutic companies since approval takes a long time, antibiotics have a short lifetime due to AMR, and distribution and usage are limited because of ethical concerns of AMR emerging ³⁰ .

Therefore, it is necessary to ask: Which alternatives to antibiotics exist?

Antimicrobial Peptides as an Alternative to Antibiotics

Antimicrobial peptides (AMPs), also called host-defense peptides, are a potential alternative to conventional antibiotics. AMPs are a diverse group consisting mostly of cationic and amphipathic peptides ranging from 10 to 50 amino acids (aa) long ¹⁷ . They are part of the innate immune defense of most eukaryotes, like animals, plants, and fungi, as well as part of the defense mechanisms of many prokaryotes ( figure 2 ). For example, frog skin is one of the richest sources of AMPs accounting for about 50 % of the currently known AMPs from animals ²⁷ . In mammals, the two most abundant classes of AMPs are defensins, exhibiting a beta-sheet structure stabilized by disulfide-bridges and cathelicidins, exhibiting a linear alpha-helical structure ²⁰ . An example of this is the human cathelicidin LL-37, which will be discussed in detail later on. Bacterial AMPs can be divided into ribosomally and non-ribosomally synthesized peptides. Polymyxins represent a class of lipopeptides synthesized by special nonribosomal peptide synthetases, which are used nowadays as a last-line defense against multidrug-resistant gram-negative bacteria ²⁵ . Bacterial ribosomally synthesized peptides are also called bacteriocins and can be classified in class I bacteriocins, which exhibit post-translational modifications like the formation of disulfide bridges or circularization, and class II bacteriocins missing post-translational modifications ¹³ . For example, lantibiotics like nisin produced by the gram-positive Lactococcus lactis are belonging to class I bacteriocins ³² . Often not classified as AMPs but rather as antimicrobial proteins are colicins. They occur in certain Escherichia coli (E. coli) strains as well as other gram-negative bacteria and are specifically active against other strains of the same gram-negative bacterial species ³⁷ . Thionins and snakins are examples of classes of AMPs originating in plants ²⁰ .

The mode of action of most AMPs involves disruption of cell membranes ( figure 2 ). This can be facilitated by different mechanisms, depending on the structure and charge distribution of the AMP, described either by the toroidal pore model, the barrel-stave model, or the carpet-like model ²⁰ . In all three models, the AMP binds to or is inserted into the cell membrane either as a monomer or in a multimeric form. This leads to either collapse of the lipid bilayer or pore formation, disrupting the membrane potential and ultimately to cell lysis ¹² . This binding mechanism to cell membranes is facilitated by the cationic and amphiphilic nature of most AMPs. The cationic moiety of the AMP is associated with anionic phospholipid headgroups, while the hydrophobic moiety is inserted into the hydrophobic membrane core ¹² . For instance, the human cathelicidin LL-37 acts via the carpet-model, it binds parallel to the cell membrane and mostly interacts with negatively charged phospholipid headgroups by its positively charged residues, which are located on one face of its alpha-helix ¹⁶ . Apart from disrupting cell membranes, AMPs have also been found to act intracellularly on different targets, for example by inhibiting DNA synthesis, protein synthesis, protein folding, or cell wall synthesis ⁵ . This is thought to be relevant especially at lower AMP concentrations too low for the AMP to cause rupture of the cell membrane, but where the AMPs might enter target cells by the same mode of action of binding and transiently permeabilizing the cell membrane ³¹ . In fact, acting on intracellular targets might be the main killing mechanism of many AMPs ^1, ³¹ .

Despite their broad action on cell membranes, many AMPs have a target specificity towards certain classes of organisms. The majority of the AMPs known today are active against bacteria, more precisely acting on the bacterial inner membrane ²⁰ . This is because the bacterial inner membrane contains negatively charged phospholipids like phosphatidylethanolamine, phosphatidylglycerol and cardiolipin, and exhibits a net negative charge ¹² . In contrast, mammalian cell membranes are rather neutrally charged, which explains the lower activity of AMPs towards them ¹² . Furthermore, the positively charged AMPs are associated with negatively charged lipopolysaccharides, and teichoic acids. These are located on the outside of gram-negative and gram-positive bacterial cells, respectively, which promotes AMPs' binding to the inner cell membrane ²⁰ . Besides anti-bacterial peptides, AMPs can also be active against fungi, viruses like HIV, parasites, and cancer cells ²⁰ .

The above-described AMPs' mode of action rises an important question: Which advantages do AMPs have compared to antibiotics? Since their action against bacteria relies on similar targets as those of antibiotics, one might fear the occurrence of antimicrobial resistance (AMR) towards AMPs. Indeed, bacteria have evolved several resistance mechanisms towards AMPs, like altering the phospholipid composition of their cell membrane, modifying molecules in their cell wall, or the excretion of proteases to degrade AMPs ¹ . However, the situation is not the same as with antibiotics. On the one hand, the bacterial resistance genes are located in the bacterial genome, not on plasmids, as it is the case for antibiotic resistance genes like beta-lactamases described earlier ¹ . Therefore, resistances against AMPs are unlikely to be conducted via horizontal gene transfer, which limits their spread. On the other hand, AMPs' mode of action not only relies on the direct killing of bacteria but their additional immunomodulatory properties as well ⁵ . They stimulate the innate immune system, which leads to indirect actions against pathogens ⁶ . Therefore, AMPs are less likely to lose their in vivo antibacterial activity than antibiotics ¹ .

Figure 2: Antimicrobial peptides (AMPs) and their applications in iGEM. [A] Many different AMPs originating from animals, plants, and bacteria have been used by iGEM teams to solve real-life problems. The most common project ideas include the expression of one or multiple AMPs in a bacterial chassis. These engineered strains were either meant to be applied directly as a probiotic, or the expressed AMPs were meant to be purified and applied as a peptide drug; [B] Different modes of action of AMPs ⁴⁸ ; Created with BioRender.com

Challenges when Working with AMPs

Up till now, 3273 AMPs are registered in the Antimicrobial Peptide Database ⁴⁹ , from which many are poorly characterized. This holds great potential for biotechnology or synthetic biology. Since AMPs are peptides, they can be easily modified, linked to various molecules for coating, and different expression and production platforms may be developed, just to name a few possibilities. This is likely to be one of the reasons for the large number of iGEM teams, which have been working with AMPs in the past ( figure 2 ) ⁴⁸ .

Regarding peptide expression, one of the challenges, which has been addressed by several iGEM and other research projects, is the toxicity of AMPs towards bacteria like E. coli , which is one of the most common hosts for protein expression. Solutions involve the expression of AMPs in E. coli with cleavable fusion proteins for charge neutralization ²² or expression of AMPs as polymers with aggregation seeds leading to the formation of inclusion bodies to burrow the AMPs' charge within ³⁵ . Another approach is to use expression hosts invulnerable to AMPs, which for instance has been demonstrated for the yeast Pichia pastoris ¹⁹ .

When considering AMPs as possible alternatives to antibiotics, it is also important to consider potential side effects on the human body. AMPs' mode of action on cell membranes makes it more difficult to predict their activity towards mammalian cells compared to antibiotics, which often have targets unique to prokaryotes. Therefore, it can be a deception to consider all membrane disrupting peptides specifically to be antimicrobial ³⁹ . Possible hemolytic or cytotoxic activities of AMPs are important to consider when working with or further developing an AMP ¹⁶ . Often, fragments of an AMP may exist, which exhibit a similar antibacterial but a reduced activity against other cell types. This is for example the case for KR-12, which was found to be the smallest antibacterial fragment of the human cathelicidin LL-37 ¹⁶ , as well as for the fragment [R4,R10]C1b(3–13) (CHEN) of the AMP chensinin-1b ⁵⁰ .

One of the greatest drawbacks in researching AMPs as antibacterial drugs is their poor in vivo stability ⁴⁰ . This is mostly due to proteolysis or heat inactivation in vivo ¹⁴ . Cyclization of a peptide's backbone, as well as the addition of disulfide bridges, can increase its exoprotease and heat stability ²⁴ . For instance, fragments of the human cathelicidin LL-37 have been backbone cyclized and dimerized by peptide organic synthesis, leading to increased serum stability ¹⁶ . Based on this principle, iGEM Team Heidelberg developed a toolbox to cyclize proteins in biological expression hosts, based on split-inteins ²¹ . Inteins are autocatalytic protein splicing domains, which occur naturally and have been engineered for several applications in protein engineering ²⁸ . Other possibilities to cyclize proteins involve enzyme-like sortases ⁴⁷ or cyclizing asparaginyl endopeptidases ⁴⁵ . Regarding the stabilization of AMPs, Koehbach et al. recently demonstrated an approach involving plant cyclotides ²⁶ . Cyclotides are highly stable circular peptides, which comprise around 30 amino acids folded in a compact cyclic cystine knot (CCK) motif stabilized by three disulfide bonds ¹⁴ . In between its six cysteine residues, cyclotides contain so-called loops, which have low aa sequence conservation and can therefore be replaced – a process called grafting – with arbitrary peptide sequences ¹⁵ . This allows the combination of the stable cyclotide scaffold with the bioactivity of a grafted peptide, as has been demonstrated for the AMP optP7, leading to increased serum stability albeit attenuating the AMPs' antibacterial activity ²⁶ .

Our Project: ATHELAS

In our iGEM project, we tried to implement the cyclotide-AMP grafting approach followed by Koehbach et al. in vivo, by expressing grafting constructs in plant species Nicotiana benthamiana (N. benthamiana) . By doing so, we have developed a screening platform for stabilized antimicrobial peptides, called ATHELAS: AMP-thioknot herbal expression and low-effort activity screening. We chose to work with N. benthamiana since apart from other eukaryotes like yeast, plants are a promising expression host for AMPs: They are likely to produce correctly folded AMPs originating from eukaryotes, they are able to perform post-translational modifications, and the production of AMPs in plants is 10 to 50 times cheaper compared to E. coli ⁴⁶ . It was also shown that plants' own microbiome is not adversely affected by recombinant expression of AMPs ⁴¹ .

Construct Design

AMPs to be stabilized enter our screening system in a theoretical design phase, starting by choosing peptides appropriate to our grafting approach: AMPs should be as small as possible, preferably not exceeding a length of 12 aa, and their secondary structure should be as simple as possible. This is because cyclotide loops, which are the location of grafting, are quite rigid due to their location in the stable CCK network and comprise no more than eight aa residues ¹⁴ . Another desired characteristic for the AMPs is low hemolytic and cytotoxic activity. To implement and test our screening system, we chose the two short, alpha-helical AMPs KR-12 and CHEN described earlier. Next, the sequence of the whole grafting construct was designed. As a cyclotide scaffold, we decided to use the trypsin inhibitor cyclotide MCoTI-II, because it lacks antimicrobial or cytotoxic activity itself ⁵¹ and was successfully used in grafting approaches before ¹⁵ . We designed constructs with either one or two identical or different AMPs grafted in loop 1 and/or loop 6, which seem to be the biggest and most flexible loops in MCoTI-II. Also, we included glycine-serine (GS) linkers in between grafted AMP and cyclotide backbone, to give the AMPs more flexibility ¹⁰ . Finally, our constructs contain a His6 peptide tag in loop 5, which can be used for affinity purification and detection later on. To test whether the designed constructs are in principle sterically feasible, we used several platforms to predict their three-dimensional structure.

Modeling

The grafting procedure bears many uncertainties, for instance, the grafted AMP may lose its activity in the novel construct ²⁶ . To select promising construct designs, and to get a rough idea of the grafting constructs' structure and interaction with biological membranes, we used several bioinformatics tools. Molecular dynamics simulations and molecular docking yield insights into the interaction of our constructs with phospholipid bilayers of different compositions. These Drylab projects were subject of our partnership with iGEM team Kolkata , and of our collaboration with iGEM team CCU Taiwan .

For more detail see our results page.

Cloning

Following construct design, the optimized constructs enter the actual Wetlab phase, which starts with cloning vectors for the expression in N. benthamiana . One of the desired features of our ATHELAS system is that it should allow fast and flexible production of new constructs. Therefore, we designed a fast and highly efficient cloning schedule based on Golden Gate assembly vectors for protein expression in plants ³ . After ordering the newly designed construct sequences by DNA synthesis, cloning them into level I vectors, and confirming their sequence by Sanger sequencing, it only takes one cloning step to bring the construct's sequence into the final N. benthamiana expression vector. Due to blue-white selection and the high efficiency of Golden Gate cloning, constructs can be yielded in only one week. Besides the actual grafted construct, which gets embedded into a cyclotide precursor sequence (Oak1 precursor), the expression vector contains two additional genes: GFP, which acts as a reporter gene and allows monitoring of the transfection success in N. benthamiana later on, and the cyclizing asparaginyl endopeptidase CtAEP1. This enzyme recognizes the precursor and cyclizes the grafting construct in vivo, as has been demonstrated for other cyclotides before ³⁴ .

For more detail see our results page.

Expression

After an expression vector is obtained and verified, the desired cyclic AMP construct is expressed in N. benthamiana transiently. Therefore, agrobacteria-mediated transfection by agroinfiltration is used. After transforming agrobacteria with the expression vectors, the bacteria are used to infiltrate N. benthamiana leaves together with a p19 suppressor of gene silencing agrobacterial strain, which enhances protein yield significantly ⁴ . Compared to stable transfection of plants, transient transfection does not yield uniform and stable genetically modified plant lines but is much faster and therefore ideal for our screening platform ³⁴ . Transfected plants are incubated for three days before the infiltrated leaves can be harvested and further processed. The co-expressed GFP is thereby used to control the extent of successful transfection in the plant's leaves.

For more detail see our results page.

Extraction, Purification, and Analytics

To obtain our expressed cyclic constructs from the harvested N. benthamiana leaves, whole crude leaf tissue extracts are first obtained by homogenizing and extracting the homogenized tissue. In this step, we tested different extraction buffers and extraction methods in order to optimize the extraction of our constructs from plant tissue and to maximize the yield. The extracts were then either used for His tag affinity purification, with the goal to achieve highly purified AMP constructs or directly used for further steps. In the following analytical phase, we obtained results on our peptide yield, which allowed us to draw conclusions and optimize our upstream methods. Our analytical methods included western blotting and MALDI-TOF MS.

For more detail see our results page.

Activity Assessment

The final phase of our ATHELAS screening system is testing the antibacterial activity. Here, affinity-purified samples as well as crude plant extracts were tested, to obtain further data on the success of our extraction and purification protocols. To compare the antibacterial activity of our grafted constructs to the activity of their linear ancestors, we ordered chemically synthesized KR-12 and CHEN AMPs as positive controls. In order to maintain the safety of our team members, we decided to use non-pathogenic E. coli and B. subtilis strains in our activity tests, since they represent both members of gram-negative and gram-positive bacteria, respectively. We tested antibacterial activities in different assays and different media obtaining as much information on our constructs as possible.

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About Us

We are the iGEM Team Tuebingen, a group of motivated students who are working on creating a fast screening platform for stabilized peptides. We are aiming to provide a system that gives everyone the ability to stabilize peptides such as antimicrobial peptides to create better medical agents.